Project 1: Trafficking and posttranslational modification of the Notch receptor. We have been studying the role of trafficking and posttranslational modification of the Notch receptor protein for several years. The Notch signaling pathway controls the differentiation of many different cell types and tissues, and malfunction of this pathway has been shown to cause T-cell leukemia and other diseases in humans. Recently we have identified a mutant which alters the endocytic internalization of Notch, causing Notch to accumulate in enlarged endosomal vesicles. The mutation affects a gene encoding a channel-related protein, and we are currently investigating the molecular and biochemical properties of this protein. Preliminary data indicate that chemical inhibitors of this class of channel proteins impair the proteolytic activation of the Notch receptor in Drosophila cultured cells, suggesting a role for the new factor in this step of the signaling pathway. Cell biological studies in mutant Drosophila tissue clones together with genetic and biochemical analyses of tagged Notch pathway components are being used to investigate the function of this new channel-related protein in the signaling pathway.- Project 2: Analysis of a gene family in Drosophila related to mammalian beta-amyloid binding protein. Numerous studies in recent years have uncovered a multiprotein complex termed gamma-secretase that has a conserved proteolytic function in both Notch signaling and amyloid peptide production in Alzheimer's disease. Both Notch and Amyloid Precursor Protein (APP), from which amyloid peptide is derived, are type 1 single-pass integral membrane proteins that are substrates for gamma-secretase-mediated intramembrane proteolysis. The Drosophila genome encodes three proteins related to a mammalian protein known to bind amyloid, and one of these fly proteins is thought to participate in Notch signaling-related developmental processes. These observations prompted us to initiate a genetic and molecular characterization of this gene family with the aim of elucidating its potential function(s) in Notch biosynthesis, trafficking, activation, degradation, or other aspects of receptor metabolism. A combination of RNA interference (RNAi), biochemical, and confocal microscopy approaches in cultured cells and transgenic flies are being utilized in this project.